For immunoassay, various methods have been proposed and practiced such as an radioactive immunoassay, and an enzyme antibody method. For example a radio immunoassy (RIA) or immunoradiometric assay (IRMA) utilizes a marking of a competitive antigen or an antibody with a radioactive nucleus and quantitatively determines the antigen by a measurement of specific radioactivity. This method is advantageous in a high sensitivity, but is associated with the safety of the radioactive nuclei and necessitates a facility and apparatuses exclusive for the assay. Also the anzyme antibody method utilizing an enzyme for the antibody marking is easier in handling in comparison with the radioactive immunoassay and has a practical sensitivity, but further improvements in the sensitivity and in the easy of handling are being desired.
On the other hand, a method of utilizing a magnetoresistive film for detecting a magnetic particle coupled with a target substance thereby easily detecting the target substance is proposed by D. L. Graham et al., Biosensors & Bioelectronics 18, 483(2003) (reference 1). The reference 1 utilizes two GMR (giant magnetic resistance effect) film of a size of 2×6 μm for detecting a magnetic particle of a diameter of 2 μm. Biotin is bonded to the surface of a GMR film for enabling fixation of the magnetic particle, but is not bonded to the other GMR film. Also the magnetic particle is modified with avidin.
Since avidin and biotin are bonded very strongly, the magnetic particle is fixed on a GMR film but not on the other. The GMR film on which the magnetic particle is influenced by a floating magnetic field from the magnetic particle, and shows a resistance different from that of the GMR film on which the magnetic particle is not fixed. The GMR film is basically constituted of a multi-layered film structure including two magnetic films and a non-magnetic metal film positioned therebetween. Its resistance depends on the relative directions of magnetization of the two magnetic films, and is relatively small when the directions of magnetization are parallel but relatively large when the directions of magnetization are antiparallel. In order to realize magnetizations of parallel and antiparallel, one of the two magnetic films constituting the GMR film is fixed in the direction of magnetization, while the other is formed by a magnetic material of a coercive force capable of a magnetic inversion by the floating magnetic field from the magnetic particle.
When a magnetic field is applied to the magnetic particle and the GMR film in a longitudinal direction thereof, thereby directing the magnetization of the magnetic particle in a direction of the applied magnetic field, a floating magnetic field 904 generated from the magnetic particle 1001 is, as shown in FIG. 9, applied to the GMR film 905 in a direction opposite to that of the external magnetic field 903. Therefore, magnetizations 902 by the two magnetic films of the GMR film 905 do not become parallel in the vicinity of the magnetic particle 901. On the other hand, the magnetic films of the GMR film 905 on which the magnetic particle is not fixed, not being influenced by the floating magnetic field 904, show parallel magnetizations over the entire film. Thus the two GMR film 905 shows different states of magnetization, thereby generating a difference in the resistance and enabling detection of the magnetic particle.
As described above, the method of detecting the magnetic particle with the GMR film is based on magnetizing the magnetic particle in a desired direction and changing the direction of magnetization of the magnetoresistive film by the floating magnetic field generated from the magnetic particle, and can easily detect the magnetic particle.
Also G. Li et al., J. Appl. Phys. 93, 7557(2003) (reference 2) discloses a sensor prepared by forming a GMR film capable of fixing a magnetic particle and a GMR film incapable of fixing a magnetic particle on a silicon wafer, and constructing a Wheatstone bridge with these GMR films and two other resistors, in which a detection signal of a magnetic particle is supplied through a sensing amplifier to a locking amplifier.
The reference 1 describes that a detection signal of the magnetic particles utilizing a single GMR film varies depending on the number of magnetic particles. This is because an area of the GMR film influenced by the floating magnetic field from the magnetic particles varies by the number of the magnetic particles. In case the area influenced by the floating magnetic field from the magnetic particles represents a very small ratio with respect to the entire area of the GMR film, the detection signal becomes very weak, whereby the magnetic particles become undetectable. According to the reference 1, for a detection current of 8 mA, a detection of a magnetic particle of a diameter of 2 μm with a GMR film of 2×6 μm provides a detection signal of 400 μV or less, whereby the two GMR films provides a resistance difference of 0.05Ω or less. As will be apparent from these experimental results, a GMR film can only detect one to several magnetic particles at maximum. Therefore the detection system described in reference 1 is incapable of detecting the magnetic particles over a wide range.
The reference 2 discloses a configuration in which plural GMR films are connected in parallel, of which a half is covered by a photoresist film and is therefore incapable of fixing the magnetic particles on the surface. Such configuration, though capable of detecting the magnetic particles over a wide range, is associated with a drawback that wirings and, peripheral circuits require a large area. It also involves a drawback that the GMR films cannot be arranged densely because of a complicated wiring arrangement, thereby increasing the entire detection area.